This invention relates to a translational reporter system. More particularly, the invention relates to a reporter system and method of use thereof for quantification of translational recoding, reinitiation, and internal initiation in eukaryotes in vivo and in vitro.
There are a number of cases where a single messenger RNA (mRNA) is translated into more than one protein by "recoding." "Recoding" has been defined as a phenomenon where the rules for translation decoding are temporarily altered through specific sites and signals built into the mRNA sequences (I. Brierly, Ribosomal Frameshifting on Viral RNAs, 76 J. Gen. Virol. 1885-1892 (1995); R. F. Gesteland & J. Atkins, Recoding: Dynamic Reprogramming of Translation, 65 Annu. Rev. Biochem. 741-768 (1996)). In some cases of recoding, special signals are far distant 3' on the message (M. J. Berry et al., Functional Characterization of the Eukaryotic SECIS Elements which Direct Seleno-cysteine Insertion at UGA Codons, 12 EMBO J. 3315-3322 (1993); W. A. Miller et al., New Punctuation for the Genetic Code: Luteovirus Gene Expression, 8 Sem. Virol. 3-13 (1997)). For studying the great majority of known cases of recoding, where the signals are close to the recoding site, it is helpful to measure the synthesis of two proteins. This has typically been done by monitoring radioactive proteins separated by SDS gel electrophoresis or by some combination with enzyme assays. These studies would be aided by a convenient and rapid assay suitable for both in vitro and in vivo screening of products.
In mammalian cells, three kinds of recoding have been described. First, redefinition of stop codons to sense codons (i.e., readthrough) allows synthesis of selenocysteine-containing proteins (A. Bock et al., Selenoprotein Synthesis: An Expansion of the Genetic Code, 16 Trends Biochem. Sci. 463-467 (1991); S. C. Low & M. J. Berry, Knowing When Not to Stop: Selenocysteine Incorporation in Eukaryotes, 21 Trends Biochem. Sci. 203-208 (1996)) and synthesis of elongated proteins in many RNA viruses, such as Moloney murine leukemia virus (MuLV) (Y. Yoshinaka et al., Murine Leukemia Virus Protease Is Encoded by the Gag-Pol Gene and Is Synthesized through Suppression of an Amber Termination Codon, 82 Proc. Nat'l Acad. Sci. USA 1618-1622 (1985)). Second, +1 frameshifting regulates expression of ornithine decarboxylase antizyme. The system is autoregulatory and depends on the concentration of polyamines (S. Hayashi et al., Ornithine Decarboxylase Antizyme: A Novel Type of Regulatory Protein, 21 Trends Biochem. Sci. 27-30 (1996)). Third, -1 frameshifting is used to synthesize the GagPol precursor polyprotein in retroviruses that have gag, (pro), and pol genes in different reading frames (except spumaretroviruses, J. Enssle et al., Foamy Virus Reverse Transcriptase Is Expressed Independently from the Gag Protein, 93 Proc. Nat'l Acad. Sci. USA 4137-4141 (1996)). Examples are the mouse mammary tumor virus (MMTV) gag-pro frameshift (T. Jacks et al., Two Efficient Ribosomal Frameshifting Events Are Required for Synthesis of Mouse Mammary Tumor Virus Gag-related Polyproteins, 84 Proc. Nat'l Acad. Sci. USA 4298-4302 (1987); R. Moore et al., Complete Nucleotide Sequence of a Milk-transmitted Mouse Mammary Tumor Virus: Two Frameshift Suppression Events Are Required for Translation of Gag and Pol, 61 J. Virol. 480-490 (1987)) and the human immunodeficiency virus type 1 (HIV-1) gag-pol frameshift (N. T. Parkin et al., Human Immunodeficiency Virus Type 1 Gag-Pol Frameshifting Is Dependent on Downstream MRNA Secondary Structure: Demonstration by Expression In Vivo, 66 J. Virol. 5147-5151 (1992)).
Continuous efforts to study the elements on messenger RNAs that signal recoding have led to the development of several reporter systems. In some studies, the efficiency of recoding is assessed by analysis of .sup.35 S-met-labeled translation products. Constructs direct ribosomes to initiate in the zero frame to translate open reading frame-1 (ORF-1). Before the termination of ORF-1, however, a recoding signal in a test sequence directs a proportion of ribosomes to bypass the terminator. In some systems, this occurs by shifting the reading frame; in others, an amino acid is inserted for the stop codon. In both cases, the chimeric product has the upstream reporter fused to the downstream reporter. After electrophoresis of the translation products on an SDS polyacrylamide gel, the ratio of the shorter, upstream product to the chimera reflects the recoding efficiency of the test sequence. Several enzymatic reporter assays have been developed for in vivo studies using chloramphenicol acetyl transferase (cat) (R. Martin et al., Aminoglycoside Suppression at UAG, UAA and UGA Codons in Escherichia coli and Human Tissue Culture Cells, 217 Mol. Gen. Genet. 411-418 (1989)) or firefly luciferase (M. Cassan et al., Expression Vectors for Quantitating In Vivo Translational Ambiguity: Their Potential Use to Analyse Frameshifting at the HIV Gag-Pol Junction, 141 Res. Virol. 597-610 (1990); H. Reil & H. Hauser, Test System for Determination of HIV-1 Frameshifting Efficiency in Animal Cells, 1050 Biochim. Biophys. Acta 288-292 (1990)). If the test sequence contains a frameshift signal, its control construct contains a one-base insertion or deletion (in-frame control). If the signal specifies readthrough, in the control, the stop codon is changed into a sense codon by a one-base substitution. Transfection efficiencies are determined by comparison with lacZ gene product from a cotransfected construct.
To obtain a direct measure of efficiency, in two cases (H. Reil et al., A Heptanucleotide Sequence Mediates Ribosomal Frameshifting in Mammalian Cells, 67 J. Virol. 5579-5584 (1993); G. Stahl et al., Versatile Vectors to Study Recoding: Conservation of Rules between Yeast and Mammalian Cells, 23 Nucleic Acids Res. 1557-1560 (1995)) a .beta.-galactosidase reporter gene was inserted in the upstream ORF frame with firefly luciferase downstream providing an internal control for initiating ribosomes. This removed the issue of monitoring transfection efficiencies. The advantage of an activity-based reporter system is that it allows estimation of the ratio between upstream and downstream reporters for the control construct, whereas protein gels only show one product, corresponding to the fusion product. The activity ratio of the positive control can be used to normalize values obtained from the corresponding recoding signal construct. The power of this system, when applied systematically to each construct, is demonstrated in a recent report that showed that the efficiency of HIV-1 frameshifting is directly related to the stability of the stem loop (L. Bidou et al., In Vivo HIV-1 Frameshifting Efficiency Is Directly Related to the Stability of the Stem-Loop Stimulatory Signal, 3 RNA 1153-1158 (1997)). Although .beta.-galactosidase reporters are useful in vivo, they are not suitable for in vitro translation due to the length of the lacZ coding sequence (3 kb).
In view of the foregoing, it will be appreciated that providing a reporter system for in vivo and in vitro measuring translation coupling efficiency of recoding mechanisms such as frameshifting and readthrough would be a significant advancement in the art.